A COMPLEX ARCHITECTURE FOR GENETIC MODIFICATION OF CARDIOVASCULAR PHENOTYPES IN MOUSE MODELS OF TGF-BETA VASCULOPATHIES

TGFβ;Loeys-Dietz Syndrome;Developmental Genetics;Gene Mapping;Persistent Truncus Arteriosus;Human Genetics and Molecular Biology

Calderon Giadrosic, Juan FranciscoAvramopoulos, Dimitrios G. ;

Johns Hopkins University

关键词: TGFβ;    Loeys-Dietz Syndrome;    Developmental Genetics;    Gene Mapping;    Persistent Truncus Arteriosus;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/40651/CALDERONGIADROSIC-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Our work on mouse models of Loeys-Dietz syndrome (LDS) has shown that these mice recapitulate human disease and develop aortic root aneurysm, which is associated with a signature of increased transforming growth factor  (TGFβ) signaling. As in Marfan syndrome (MFS), aortic disease in LDS mouse models is sensitive to and fully reversed by the action of losartan, an angiotensin receptor blocker (ARB) that blunts TGFβ signaling, highlighting the molecular overlap between these two diseases.The instrumental role that mouse models of both MFS and LDS have played in furthering our knowledge of the pathophysiology of these diseases illustrates the importance of animal models for the study of human genetic disorders. However, the realization that the genetic background of an animal model can modulate the clinical expression of disease-specific phenotypic features has also highlighted the need for understanding the role of natural genetic variation in phenotypic variability.The research presented in this dissertation is based on the observation that in the context of LDS, there is great phenotypic variability associated with specific mouse strain backgrounds. We went on to prove that while postnatal aneurysm progression, which is associated with excessive TGFβ signaling, is blunted upon crossing LDS mutations onto a C57BL/6J (B6) background, LDS mice on this background also show high penetrance of perinatal death due to persistent truncus arteriosus with interrupted aortic arch (PTA/IAA), a congenital heart defect previously associated with insufficient TGFβ signaling in the cardiac neural crest. To assess the effect of the B6 background on the phenotype we performed a test of dominance. A single cross of Tgfbr2G357W/+ mice congenic on a 129S6/SvEvTac (129SvE) background to B6 resulted in 67% of mutant pups with PTA/IAA, while the control backcross onto 129SvE only resulted in 3.7% (n=1/27) of mutant pups with PTA/IAA (p<1E-9, Fisher;;s exact test). In the context of this mutation, we concluded that the B6 background has a major dominant effect with incomplete penetrance on the generation of PTA/IAA, which could be due to other loci that act in a recessive or dosage-dependent manner. Interestingly, a test of dominance performed with Tgfbr1M318R/+mice yielded complete absence of outflow tract defects, leading to the conclusion that in this scenario, the B6 background had a recessive effect on the generation of PTA/IAA, which was confirmed when a second backcross onto this background was performed and 50% of the mutant pups showed PTA/IAA.Since PTA is introduced when LDS mutations are bred onto a mixed background, we used this discrete phenotype to map the relevant modifier alleles. Tgfbr2G357W/+ mice on a pure 129SvE background were bred to F2 WT mice with extensive recombination between B6 and 129SvE chromosomes. The resulting E17.5 fetuses were phenotyped for TA/IAA and DNA from the phenotyped embryos was collected and genotyped using a SNP array specially designed for mouse intercrosses and linkage studies.A genome-wide analysis revealed a single major B6-specific locus associated with TA/IAA on mouse chromosome 9, with a -log10(p)=9 at a map position coincident with the Tgfbr2 gene. A minor linkage signal was also apparent on chromosome X with a -log10(p)=2.75.Tgfbr2 emerged as a promising candidate modifier gene on chromosome 9, both by virtue of its known function and the presence of strain-specific variation in the 3;;UTR. We recognized that variation in the 3;;UTR can affect regulation of gene expression and went on to test for potential strain-specific Tgfbr2 expression differences. In addition, an unbiased search for microRNAs (miRNAs or miRs) that target Tgfbr2 revealed that miR-20b and miR-106a were potential candidates because they are encoded within the suggestive association peak on chromosome X. A luciferase reporter allele harboring the B6 Tgfbr2 3;;UTR showed dramatically reduced expression when compared to its 129SvE counterpart (p<1E-9). Moreover, luciferase activity was equalized between the reporter alleles with strain-specific 3’UTRs upon addition of a miR-106a or miR-20b antagonist. Taken together, these data suggest that strain-specific differences in Tgfbr2 expression are fully accounted for by variable susceptibility to miR-mediated suppression of translation. To further understand if these changes in gene regulation affect the levels of TβRII protein on the surface of cells in the arterial wall and, subsequently, TGF activity, we isolated vascular smooth muscle cells (VSMCs) from WT B6 and 129SvE animals and stimulated them with low concentrations of TGFβ1 ligand. Surface levels of TβRII and intracellular levels of phosphorylated SMAD2 (pSMAD2; the major readout of TGFβ pathway activity) were measured by flow cytometry. Higher levels of TβRII and intracellular pSMAD2 were observed VSMCs isolated from 129SvE mice.While the severity of postnatal phenotypes driven by excessive TGFβ signaling, such as aneurysm progression, is significantly ameliorated on a B6 background when compared to 129SvE in the context of a LDS mutation, the development of prenatal phenotypes associated with insufficient TGFβ levels is greatly accentuated. We provide evidence for a complex architecture of background-specific phenotypic modification in LDS and perhaps other TGFβ vasculopathies. We show that Tgfbr2 gene expression is influenced by the levels of microRNAs, such as miR-106a and miR-20b, as well as by the differences in accessibility of these miRs to the 3;;UTR of Tgfbr2 mRNA, which are dictated by sequence variants that distinguish B6 from 129SvE in this gene region.In summary, we present evidence that suggests a novel regulatory mechanism of TGFβ signaling in the context of LDS-causing mutations. This mechanism is associated with overt defects in heart morphogenesis (increased risk of PTA/IAA) and postnatal aortic wall homeostasis (suppression of postnatal aortic aneurysm progression).It is our strong conviction that therapeutic strategies that mimic nature’s success at attenuating postnatal vascular disease on the B6 background (prominently including TGFβ antagonism) hold strong promise for people with LDS and perhaps other TGFβ vasculopathies.